Dental plaque constitutes an ecological niche, activities of which are highly correlated with the onset and development of dental caries. Insoluble glucan, termed mutan, provides the molecular framework around which plaque accumulates. The goal of the project is to reduce the bulk of plaque by disrupting its molecular architecture. We will then determine the effect of that disruption on the prevalence of dental caries. In vitro studies have demonstrated that applications of mutanase have substantially reduced the accumulation of plaque carbohydrate. Results from studies conducted in animals and humans have repeatedly shown that topical applications of mutanase [alpha(1->3) glucan, 3-glucanohydrolase] reduce plaque formation and caries. Thus, mutanase may have promise in the prevention of dental caries. The results from studies conducted on humans showed that mutanase, presented in chewing gum, reduced the amount of plaque and gingivitis compared with placebo groups. However, in the oral environment, the enzyme had to be applied to the tooth surface too frequently to be practical. We propose a mechanism to continuously supply mutanase to the location where it would be most efficacious, that is, within dental plaque. Cloning of the mutanase gene into Streptococcus sanguis, an organism that normally colonizes the tooth surface, represents such a mechanism. In addition, the achievement of this goal would clarify the role of mutan in the formation of plaque and the pathogenesis of oral disease. Recombinant DNA techniques will be used to clone the gene for mutanase from the fungus Trichoderma harzianum. The gene will be subcloned into a novel streptococcal expression-secretion vector and introduced into S. sanguis. The genetic construction used will permit the recombinant organism to synthesize and secrete a catalytically active enzyme (mutanase). The recombinant S. sanguis will be used in studies, together with S. mutans, in an in vitro model to assess its effect on plaque accumulation and then in the rat model system to determine its effectiveness against the development of caries. These studies will result in: a) an enhanced understanding of the role of dental plaque in the pathogenesis of dental enhanced understanding of the role of dental plaque in the pathogenesis of dental caries and information that will aid in the prevention of caries; b) an understanding of streptococcal promoters and signal sequences required for secretion of foreign gene products; c) an increased knowledge of heterologous DNA stability in the oral streptococci; and d) a novel method for the prevention of caries may be developed. The completion of this project will establish important new principles for the application of molecular biology to the prevention and treatment of oral disease.